Proteins are an important component of the cellular machinery and their function highly correlates to structure and dynamics. Combining ion mobility, native mass spectrometry (IM/MS) and gas-phase ion/ion reactions provides a sensitive and relatively quick approach to study protein structure. Unlike solution phase labeling, gas-phase ion/ion reactions are achieved in-line under controlled conditions to improve reaction efficiency and resulting products are easily separated by IM and analyzed by tandem MS. In this study, all cross-linking reagents contained two sulfonate groups, facilitating electrostatic attachment to accessible protonated residues to form a product analyzed by tandem MS. One scaffold type ‘A’ included phenyl ring(s) with sulfonates attached while another type ‘B’ consisted of a more flexible spacer linker between two sulfonated N-hydroxysuccinimide (sulfo-NHS) ester reactive groups. Native protein (cytochrome c or α-synuclein) and −2 sulfonated ions reacted in the trap cell with the reactant and charge-reduced product ions separated by ion mobility, followed by electron-capture dissociation (ECD). The cross-linking reagents of various spacer lengths and flexibility resulted in different cross-linked residue pairs, thus providing detailed residue-level information pertaining to protein folding and dynamics. Additionally, the modified sites were used as distance constraints to model the structure of cytochrome c and α-synuclein by molecular dynamics (MD). Overall, the results support the application of a gas phase ion/ion cross-linking strategy combined with IM/MS/MS and MD simulation to provide structural insights into protein conformations.

Probing protein structure via gas-phase ion/ion cross-linking coupled to on mobility/time-of-flight mass spectrometry

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